Genetically encoded sensors are very useful for imaging small molecules in living cells in order to detect them. The modular and programmable nature of nucleic acids, coupled with their capacity for molecular recognition, positions nucleic acids as a uniquely versatile scaffold for molecular sensing within the cellular milieu.
Cyclic adenosine monophosphate (3′-5′-cyclic adenosine monophosphate, cAMP) is a secondary messenger and is important in many biological processes. Thus, it becomes very important to detect and quantify cAMP in biological systems. Presently radio-immunoassays are being used for measuring cAMP in in vitro studies which are variable, costly and highly time consuming and the existing sensors for live cell imaging of cAMP are based on proteins which are natural intracellular targets of cAMP and hence these sensors may interfere with cellular physiology.
Modular RNA aptamer sensors generally known in the art have three domains: target recognition domain, communication module and reporter domain. Reporter domain comprises molecules that bind a fluorescent label, thus emitting a fluorescent signal. Target recognition domain binds the target to be detected and communication module transfers the information from target recognition domain to reporter domain such that whenever recognition domain binds to target, the reporter domain will give fluorescence signal.
Spinach is an RNA aptamer that binds the chromophore DFHBI. DFHBI, in general, is a chromophore with low fluorescence yields, i.e., it is very less fluorescent. However, when this chromophore is bound to Spinach, the fluorescence quantum yield increases by a factor of 80. This aptamer has a stem-loop called stem-loop-2, whose structure is important in order for binding to DFHBI but its sequence is not important. Stem loop-2 has been used as communication module for designing sensors for target analytes whose aptamers are known by attaching aptamer for the target analyte (which is target recognition domain) at stem loop-2.
However, RNA aptamers for various analytes have 5′ ends that need to be free for their binding action e.g. cAMP aptamer. Spinach cannot be used as reporter domain for making sensors for such analytes as attaching aptamer to loop of stem-loop-2 will make analyte aptamer lose its 5′ end. So, there is no Spinach based sensor for cAMP. Further, the available Spinach based aptameric sensors for cdiGMP, Adenosine, ADP SAM etc. are not ratiometric.
Therefore, in order to make Spinach aptamer amenable for such analytes, it is required to carry out a rearrangement in Spinach so as to create a free 5′ end near stem-2. However, it is observed in the art that rearranging of nucleic acid molecules can affect their activity.